Changes in the expression of the potassium channels TASK1, TASK3 and TRESK in a rat model of oral squamous cell carcinoma and their relation to malignancy.

OBJECTIVES: Potassium channels have been proposed to promote cancer cell proliferation and metastases. Thus, we investigated the expression pattern of three 2-pore domain potassium channels (K2Ps) TASK1, TASK3 and TRESK in advanced oral squamous cell carcinoma (OSCC), the commonest oral malignancy. DESIGN: We used 4-nitroquinoline-1-oxide (4-NQO) to induce high grade OSCC in male adult rats. We then used immunohistochemistry and Western blotting to study the distribution and expression pattern of TASK1, TASK3 and TRESK in normal versus cancerous tissue. We also examined the expression of ß-tubulin III (ß-tub3), a marker associated with resistance to taxane-based chemotherapy and poor patient prognosis, and its correlation with the K2Ps. Finally, we studied the expression of TASK1, TASK3 and TRESK in human samples of SCC of oral origin. RESULTS: We found that TASK3 was significantly up-regulated whereas TASK1 and TRESK were both significantly down-regulated in advanced, poorly differentiated OSCC. Both, rat and human SCC showed a significant increase in the expression of ß-tub3. Interestingly, the expression of the latter correlated positively and significantly with TASK3 and TRESK but not TASK1 in rat OSCC. Our initial results showed a similar pattern of up and down regulation and correlation with ß-tub3 for these three K2Ps in human SCC. CONCLUSIONS: The changes in expression and the co-localization with a marker of resistance to taxanes like ß-tub3 turn TASK1, TASK3 and TRESK into potentially new prognostic tools and possibly new therapeutic targets for OSCC.

Mosquito bloodmeal preferences in two zoological gardens in Germany.

Because they provide a high density and diversity of vertebrate species, small water pools and shaded environments, zoological gardens offer ideal living conditions for numerous mosquito species. Depending on their host preferences and vector competencies, these species may be able to transmit pathogens between native and non-adapted exotic blood host species, thereby causing morbidity and mortality among valuable zoo animals. To determine the extent to which native mosquito species feed on captive and wild animals, as well as on humans, in two German zoological gardens, mosquitoes were collected over two seasons by trapping and aspirating. A total of 405 blood-fed specimens belonging to 16 mosquito taxa were collected. Genetic bloodmeal analysis revealed 56 host species, mainly representing mammals of the zoo animal population, including exotic species previously not known as blood hosts of the mosquito species collected. These results indicate opportunistic feeding patterns with low host-specificity in the analysed mosquitoes, although these could be grouped, according to their bloodmeals, into 'amphibian-', 'non-human mammal-' and 'non-human mammal and human-' feeding species. As the blood-feeding preferences of vector-competent mosquito species are major determinants of vector capacity, information on the blood-feeding behaviour of mosquitoes in zoos is crucial to the success of targeted vector management.

Genome-wide DNA hydroxymethylation identifies potassium channels in the nucleus accumbens as discriminators of methamphetamine addiction and abstinence.

Epigenetic consequences of exposure to psychostimulants are substantial but the relationship of these changes to compulsive drug taking and abstinence is not clear. Here, we used a paradigm that helped to segregate rats that reduce or stop their methamphetamine (METH) intake (nonaddicted) from those that continue to take the drug compulsively (addicted) in the presence of footshocks. We used that model to investigate potential alterations in global DNA hydroxymethylation in the nucleus accumbens (NAc) because neuroplastic changes in the NAc may participate in the development and maintenance of drug-taking behaviors. We found that METH-addicted rats did indeed show differential DNA hydroxymethylation in comparison with both control and nonaddicted rats. Nonaddicted rats also showed differences from control rats. Differential DNA hydroxymethylation observed in addicted rats occurred mostly at intergenic sites located on long and short interspersed elements. Interestingly, differentially hydroxymethylated regions in genes encoding voltage (Kv1.1, Kv1.2, Kvb1 and Kv2.2)- and calcium (Kcnma1, Kcnn1 and Kcnn2)-gated potassium channels observed in the NAc of nonaddicted rats were accompanied by increased mRNA levels of these potassium channels when compared with mRNA expression in METH-addicted rats. These observations indicate that changes in differentially hydroxymethylated regions and increased expression of specific potassium channels in the NAc may promote abstinence from drug-taking behaviors. Thus, activation of specific subclasses of voltage- and/or calcium-gated potassium channels may provide an important approach to the beneficial treatment for METH addiction.

The invasive Asian tiger mosquito Aedes albopictus (Diptera: Culicidae) in Germany: Local reproduction and overwintering.

Within the framework of a German mosquito monitoring programme, the 'Mueckenatlas' (mosquito atlas) has been established as an instrument of citizen participation in mosquito mapping. In 2015, a strikingly large number of Aedes albopictus, which had not been considered established in Germany, was submitted. Three of six collection sites showed local reproduction, with demonstration of developmental stages over three months at two sites. The third populated site was checked only once in October. Developmental stages of Ae. albopictus were found again at these three sites in spring 2016, including one site in southeastern Germany where reproduction had already been documented in 2014. Although population genetic analyses performed on specimens collected at the latter locality in 2014 and 2015 did not provide proof for hibernation, the finding of developmental stages at this and two other very same sites as in the year before and at very early times in the season strongly suggest accomplished overwintering of Ae. albopictus in Germany. Obviously, the second extremely mild winter in Germany in a row and ongoing adaptation of Ae. albopictus to the temperate European climate allow the species to push northwards from endemic regions in the south. Due to the vector competence of Ae. albopictus for numerous pathogens, including dengue, chikungunya and Zika viruses, action should be taken immediately after the detection of local reproduction to eliminate the populations.

Smoking quit success genotype score predicts quit success and distinct patterns of developmental involvement with common addictive substances.

Genotype scores that predict relevant clinical outcomes may detect other disease features and help direct prevention efforts. We report data that validate a previously established v1.0 smoking cessation quit success genotype score and describe striking differences in the score in individuals who display differing developmental trajectories of use of common addictive substances. In a cessation study, v1.0 genotype scores predicted ability to quit with P=0.00056 and area under receiver-operating characteristic curve 0.66. About 43% vs 13% quit in the upper vs lower genotype score terciles. Latent class growth analyses of a developmentally assessed sample identified three latent classes based on substance use. Higher v1.0 scores were associated with (a) higher probabilities of participant membership in a latent class that displayed low use of common addictive substances during adolescence (P=0.0004) and (b) lower probabilities of membership in a class that reported escalating use (P=0.001). These results indicate that: (a) we have identified genetic predictors of smoking cessation success, (b) genetic influences on quit success overlap with those that influence the rate at which addictive substance use is taken up during adolescence and (c) individuals at genetic risk for both escalating use of addictive substances and poor abilities to quit may provide especially urgent focus for prevention efforts.

Essential role of glucose transporter GLUT3 for post-implantation embryonic development.

Deletion of glucose transporter gene Slc2a3 (GLUT3) has previously been reported to result in embryonic lethality. Here, we define the exact time point of growth arrest and subsequent death of the embryo. Slc2a3(-/-) morulae and blastocysts developed normally, implanted in vivo, and formed egg-cylinder-stage embryos that appeared normal until day 6.0. At day 6.5, apoptosis was detected in the ectodermal cells of Slc2a3(-/-) embryos resulting in severe disorganization and growth retardation at day 7.5 and complete loss of embryos at day 12.5. GLUT3 was detected in placental cone, in the visceral ectoderm and in the mesoderm of 7.5-day-old wild-type embryos. Our data indicate that GLUT3 is essential for the development of early post-implanted embryos.

Influence of human tryptophan hydroxylase 2 N- and C-terminus on enzymatic activity and oligomerization.

Tryptophan hydroxylase (TPH) catalyses the first and rate limiting step in the biosynthesis of the neurotransmitter serotonin. There are two TPH isoenzymes in humans, encoded by two different genes: TPH1 and the recently described TPH2. We have expressed both human enzymes and various deletion mutants of TPH2 (DeltaN44, DeltaC17, DeltaC19, DeltaC51) in COS7 cells. TPH1 and 2 displayed different kinetic properties with a lower K(m) value of TPH1. Removal of 44 amino acids from the N-terminus of TPH2 resulted in a 3-4-fold increased V(max), which indicates a strong inhibitory function of this part on the enzymes activity. TPH1 and 2 were able to form homooligomers and also heterooligomers with each other. The different deletion mutants (DeltaC17, DeltaC19 and DeltaC51), which lack the putative C-terminal leucine zipper tetramerization domain, existed as monomeric enzymes. While short deletions (DeltaC17 and DeltaC19) hardly changed V(max) values, the DeltaC51 mutant lost 99% of TPH activity. These data identify a region between the C-terminal oligomerization domain and the catalytic domain, which is indispensable for TPH2 activity.

Randomised controlled trials of homeopathy in hyperactive children: treatment procedure leads to an unconventional study design. Experience with open-label homeopathic treatment preceding the Swiss ADHD placebo controlled, randomised, double-blind, cross-over trial.

BACKGROUND: Treatment of patients with attention deficit hyperactivity disorder (ADHD) with homeopathy is difficult. The Swiss randomised, placebo controlled, cross-over trial in ADHD patients (Swiss ADHD trial) was designed with an open-label screening phase prior to the randomised controlled phase. During the screening phase, the response of each child to successive homeopathic medications was observed until the optimal medication was identified. Only children who reached a predefined level of improvement participated in the randomised, cross-over phase. Although the randomised phase revealed a significant beneficial effect of homeopathy, the cross-over caused a strong carryover effect diminishing the apparent difference between placebo and verum treatment. METHODS: This retrospective analysis explores the screening phase data with respect to the risk of failure to demonstrate a specific effect of a randomised controlled trial (RCT) with randomisation at the start of the treatment. RESULTS: During the screening phase, 84% (70/83) of the children responded to treatment and reached eligibility for the randomised trial after a median time of 5 months (range 1-18), with a median of 3 different medications (range 1-9). Thirteen children (16%) did not reach eligibility. Five months after treatment start, the difference in Conners Global Index (CGI) rating between responders and non-responders became highly significant (p = 0.0006). Improvement in CGI was much greater following the identification of the optimal medication than in the preceding suboptimal treatment period (p < 0.0001). CONCLUSIONS: Because of the necessity of identifying an optimal medication before response to treatment can be expected, randomisation at the start of treatment in an RCT of homeopathy in ADHD children has a high risk of failure to demonstrate a specific treatment effect, if the observation time is shorter than 12 months.

Noninvasive discrimination of rejection in cardiac allograft recipients using gene expression profiling.

Rejection diagnosis by endomyocardial biopsy (EMB) is invasive, expensive and variable. We investigated gene expression profiling of peripheral blood mononuclear cells (PBMC) to discriminate ISHLT grade 0 rejection (quiescence) from moderate/severe rejection (ISHLT > or = 3A). Patients were followed prospectively with blood sampling at post-transplant visits. Biopsies were graded by ISHLT criteria locally and by three independent pathologists blinded to clinical data. Known alloimmune pathways and leukocyte microarrays identified 252 candidate genes for which real-time PCR assays were developed. An 11 gene real-time PCR test was derived from a training set (n = 145 samples, 107 patients) using linear discriminant analysis (LDA), converted into a score (0-40), and validated prospectively in an independent set (n = 63 samples, 63 patients). The test distinguished biopsy-defined moderate/severe rejection from quiescence (p = 0.0018) in the validation set, and had agreement of 84% (95% CI 66% C94%) with grade ISHLT > or = 3A rejection. Patients >1 year post-transplant with scores below 30 (approximately 68% of the study population) are very unlikely to have grade > or = 3A rejection (NPV = 99.6%). Gene expression testing can detect absence of moderate/severe rejection, thus avoiding biopsy in certain clinical settings. Additional clinical experience is needed to establish the role of molecular testing for clinical event prediction and immunosuppression management.

Human cannabinoid receptor 1: 5' exons, candidate regulatory regions, polymorphisms, haplotypes and association with polysubstance abuse.

A number of lines of evidence make the gene that encodes the G-protein-coupled CB1/Cnr1 receptor a strong candidate to harbor variants that might contribute to individual differences in human addiction vulnerability. The CB1/Cnr1 receptor is the major brain site at which cannabinoid marijuana constituents are psychoactive as well as the principal brain receptor for endogenous anandamide ligands. It is densely expressed in brain circuits likely to be important for both the reward and mnemonic processes important for addiction. Altered drug effects in CB1/Cnr1 knockout mice and initial association studies also make variants at the CB1/Cnr1 locus candidates for roles in human vulnerabilities to addictions. However, many features of this gene's structure, regulation and variation remain poorly defined. This poor definition has limited the ability of previous association studies to adequately sample variation at this locus. We now report improved definition of the human CB1/Cnr1 locus and its variants. Novel exons 1-3, splice variant and candidate promoter region sequences add to the richness of the CB1/Cnr1 locus. Candidate promoter region sequences confer reporter gene expression in cells that express CB1/Cnr1. Common polymorphisms reveal patterns of linkage disequilibrium in European- and in African-American individuals. A 5' CB1/Cnr1 "TAG" haplotype displays significant allelic frequency differences between substance abusers and controls in European-American, African-American and Japanese samples. Post-mortem brain samples of heterozygous individuals contain less mRNA transcribed from the TAG alleles than from other CB1/Cnr1 haplotypes. CB1/ Cnr1 genomic variation thus appears to play roles in human addiction vulnerability.

Variation in Langerhans cell number and morphology between the upper and lower regions of the human esophageal epithelium.

Langerhans cells (LCs) are dendritic components of stratified epithelia, presenting antigens to other cells of the immune system that play a crucial role in local defense. The paucity of information about their significance in the esophageal mucosa was addressed by studying their distribution and morphology in this particular location. LCs were identified by immunohistochemical detection of CD1a, a cell-specific marker, using a monoclonal antibody, as well as by electron microscopic identification of characteristic Birbeck granules, among other typical morphological features. Cell counts carried out at 25 and 35 cm distal to the dental arch demonstrated significant differences in number and size between the two locations. The upper region contained 10.4 +/- 0.8 cells (mean +/- SEM) vs. 18.4 +/- 1.4 cells in the lower region. Also, cells in the lower region were larger and appeared to have longer dendritic processes. To our knowledge this is the first report of regional differences in number and morphology of LCs in human esophageal mucosa.

Polysubstance abuse-vulnerability genes: genome scans for association, using 1,004 subjects and 1,494 single-nucleotide polymorphisms.

Strong genetic contributions to drug abuse vulnerability are well documented, but few chromosomal locations for human drug-abuse vulnerability alleles have been confirmed. We now identify chromosomal markers whose alleles distinguish drug abusers from control individuals in each of two samples, on the basis of pooled-sample microarray and association analyses. Reproducibly positive chromosomal regions defined by these markers in conjunction with previous results were especially unlikely to have been identified by chance. Positive markers identify the alcohol dehydrogenase (ADH) locus, flank the brain-derived neurotropic factor (BDNF) locus, and mark seven other regions previously linked to vulnerability to nicotine or alcohol abuse. These data support polygenic contributions of common allelic variants to polysubstance abuse vulnerability.

Structure of the gene 2.5 protein, a single-stranded DNA binding protein encoded by bacteriophage T7.

The gene 2.5 protein (gp2.5) of bacteriophage T7 is a single-stranded DNA (ssDNA) binding protein that has essential roles in DNA replication and recombination. In addition to binding DNA, gp2.5 physically interacts with T7 DNA polymerase and T7 primase-helicase during replication to coordinate events at the replication fork. We have determined a 1.9-A crystal structure of gp2.5 and show that it has a conserved OB-fold (oligosaccharide/oligonucleotide binding fold) that is well adapted for interactions with ssDNA. Superposition of the OB-folds of gp2.5 and other ssDNA binding proteins reveals a conserved patch of aromatic residues that stack against the bases of ssDNA in the other crystal structures, suggesting that gp2.5 binds to ssDNA in a similar manner. An acidic C-terminal extension of the gp2.5 protein, which is required for dimer formation and for interactions with the T7 DNA polymerase and the primase-helicase, appears to be flexible and may act as a switch that modulates the DNA binding affinity of gp2.5.

Basecalling with LifeTrace.

A pivotal step in electrophoresis sequencing is the conversion of the raw, continuous chromatogram data into the actual sequence of discrete nucleotides, a process referred to as basecalling. We describe a novel algorithm for basecalling implemented in the program LifeTrace. Like Phred, currently the most widely used basecalling software program, LifeTrace takes processed trace data as input. It was designed to be tolerant to variable peak spacing by means of an improved peak-detection algorithm that emphasizes local chromatogram information over global properties. LifeTrace is shown to generate high-quality basecalls and reliable quality scores. It proved particularly effective when applied to MegaBACE capillary sequencing machines. In a benchmark test of 8372 dye-primer MegaBACE chromatograms, LifeTrace generated 17% fewer substitution errors, 16% fewer insertion/deletion errors, and 2.4% more aligned bases to the finished sequence than did Phred. For two sets totaling 6624 dye-terminator chromatograms, the performance improvement was 15% fewer substitution errors, 10% fewer insertion/deletion errors, and 2.1% more aligned bases. The processing time required by LifeTrace is comparable to that of Phred. The predicted quality scores were in line with observed quality scores, permitting direct use for quality clipping and in silico single nucleotide polymorphism (SNP) detection. Furthermore, we introduce a new type of quality score associated with every basecall: the gap-quality. It estimates the probability of a deletion error between the current and the following basecall. This additional quality score improves detection of single basepair deletions when used for locating potential basecalling errors during the alignment. We also describe a new protocol for benchmarking that we believe better discerns basecaller performance differences than methods previously published.

Dopaminergic mRNA expression in the intact substantia nigra of unilaterally 6-OHDA-lesioned and grafted rats: an in situ hybridization study.

The present study was performed to investigate the influence of intrastriatal fetal mesencephalic grafts on dopaminergic mRNA expression in the non-lesioned substantia nigra pars compacta of unilaterally 6-hydroxydopamine-lesioned rats. The expression of dopamine transporter mRNA, synaptic vesicular monoamine transporter mRNA and tyrosine hydroxylase mRNA was assessed in adjacent cryostat sections using in situ hybridization. Rotational behavior induced by apomorphine and amphetamine as well as hybridization of striatal sections cut at the grafting coordinates were used to prove the functional recovery and the presence of grafted cells, respectively. After grafting, the number of rotations was decreased and hybridization signals overlying cells in the grafted striatum were detected. Mean grain densities overlying labeled neurons in the substantia nigra pars compacta of grafted rats were compared to those of shamgrafted rats and revealed differential expression of dopamine transporter mRNA, whereas synaptic vesicular monoamine transporter mRNA and tyrosine hydroxylase mRNA expression showed no difference. The results will be discussed in relation to previous in vitro and in vivo studies suggesting a reduction of functional dopamine transporter molecules in the contralateral striatum.

Reversible fixation of carbon dioxide at nickel(0) centers: a route for large organometallic rings, dimers, and tetramers.

The reaction between bis(cycloocta-1,5-diene)nickel(0), carbon dioxide and benzaldehyde-N-furfurylideneimine (A) in 1,4-dioxane or THF results in the formation of the 24-membered organometallic macrocycles of the type [(A)Ni(-CH(R1)-N(R2)-COO-)]6(solv)n (R1: phenyl, R2: furfurylidene, solv: 1,4-dioxane in 1a, THF in 1b). According to the X-ray analyses, six monomeric nickelacyclic units are connected through six Ni-mu2-OCO-Ni bridges in these macrocycles. The cavities of the metallomacrocycles (diameter: 9.410(1) A in 1a, 9.250(1) A in 1b) each contain one solvent molecule. Reaction of 1b with Me3P results in the displacement of the peripheral ligands A by the phosphine to form the 24-membered organometallic macrocycle 1c. Both 1a and 1b isomerize in benzene to form the dimeric complex [(A)Ni(-CH(R1)-N(R2)-COO-)]2(solv)n (2). The X-ray crystal structure reveals that 2 consists of the same monomeric units as found in 1a and 1b. However two Ni-mu2-O-Ni bonds link the carboxylato groups. The solvent-dependent isomerization of 1b yielding 2 is a reversible reaction. Furthermore, the macrocycle 1b partially eliminates carbon dioxide above 20 degrees C, followed by elimination of half of the monodentately coordinated Schiff base ligands to form the planar tetrameric complex 6. This is also a reversible process.

Solution structural studies and low-resolution model of the Schizosaccharomyces pombe sap1 protein.

Sap1 is a DNA-binding protein involved in controlling the mating type switch in fission yeast Schizosaccharomyces pombe. In the absence of any significant sequence similarity with any structurally known protein, a variety of biophysical techniques has been used to probe the solution low-resolution structure of the sap1 protein. First, sap1 is demonstrated to be an unusually elongated dimer in solution by measuring the translational diffusion coefficient with two independent techniques: dynamic light-scattering and ultracentrifugation. Second, sequence analysis revealed the existence of a long coiled-coil region, which is responsible for dimerization. The length of the predicted coiled-coil matches estimates drawn from the hydrodynamic experimental behaviour of the molecule. In addition, the same measurements done on a shorter construct with a coiled-coil region shortened by roughly one-half confirmed the localization of the long coiled-coil region. A crude T-shape model incorporating all these information was built. Third, small-angle X-ray scattering (SAXS) of the free molecule provided additional evidence for the model. In particular, the P(r) curve strikingly demonstrates the existence of long intramolecular distances. Using a novel 3D reconstruction algorithm, a low resolution 3D model of the protein has been independently constructed that matches the SAXS experimental data. It also fits the translation diffusion coefficients measurements and agrees with the first T-shaped model. This low-resolution model has clearly biologically relevant new functional implications, suggesting that sap1 is a bifunctional protein, with the two active sites being separated by as much as 120 A; a tetrapeptide repeated four times at the C terminus of the molecule is postulated to be of utmost functional importance.

Co-evolution of proteins with their interaction partners.

The divergent evolution of proteins in cellular signaling pathways requires ligands and their receptors to co-evolve, creating new pathways when a new receptor is activated by a new ligand. However, information about the evolution of binding specificity in ligand-receptor systems is difficult to glean from sequences alone. We have used phosphoglycerate kinase (PGK), an enzyme that forms its active site between its two domains, to develop a standard for measuring the co-evolution of interacting proteins. The N-terminal and C-terminal domains of PGK form the active site at their interface and are covalently linked. Therefore, they must have co-evolved to preserve enzyme function. By building two phylogenetic trees from multiple sequence alignments of each of the two domains of PGK, we have calculated a correlation coefficient for the two trees that quantifies the co-evolution of the two domains. The correlation coefficient for the trees of the two domains of PGK is 0. 79, which establishes an upper bound for the co-evolution of a protein domain with its binding partner. The analysis is extended to ligands and their receptors, using the chemokines as a model. We show that the correlation between the chemokine ligand and receptor trees' distances is 0.57. The chemokine family of protein ligands and their G-protein coupled receptors have co-evolved so that each subgroup of chemokine ligands has a matching subgroup of chemokine receptors. The matching subfamilies of ligands and their receptors create a framework within which the ligands of orphan chemokine receptors can be more easily determined. This approach can be applied to a variety of ligand and receptor systems.

Detection of C-type natriuretic peptide in fetal circulation.

C-type natriuretic peptide (CNP) belongs to the natriuretic peptide family that plays an important role in the control of blood pressure, renal function and volume homeostasis. In contrast to the atrial natriuretic peptide and brain natriuretic peptide, CNP acts in an autocrine/paracrine fashion and is considered to be the endothelial component of the natriuretic peptide system. CNP has a high expression and tissue-specific regulation in reproductive organs. Using a radio-immunoassy for CNP-22 we measured for the first time CNP in fetal blood. Samples were taken by cordocentesis in a group of fetuses with rhesus isoimmunisation (10.74 +/- 2.81 pg/ml), fetuses with rhesus isoimmunisation after intravascular transfusion (10.03 +/- 4.01 pg/ml) and a group with structural anomalies (12.9 +/- 5.67 pg/ml). A group of healthy fetuses was used as controls (11.64 +/- 4.32 pg/ml). In contrast to ANP, the fetal CNP-plasma concentrations remain stable in the investigated fetal diseases and after volume load during intravascular transfusion. Moreover, fetal CNP-plasma levels are higher than previously measured maternal concentrations in normal pregnancies. Therefore, the fetus expresses CNP independently of the maternal circulation.